The goals of this proposal are to complete the physical mapping and phenotypic analysis of a combined set of 132 temperature sensitive vaccinia virus mutants from two different mutant collections, Dales and Condit, and to study in detail a defined subset of these mutants in order to refine our understanding of virion morphogenesis. In Aim 1, mutants from each of the unmapped or partially mapped complementation groups in the combined Dales-Condit collection of vaccinia virus temperature sensitive mutants will be mapped to a single gene using marker rescue. In Aim 2, heretofore uncharacterized representatives from each complementation group of in the combined Dales-Condit collection will be subjected to a first-level phenotypic analysis, consisting of assays for viral DNA replication, viral protein synthesis, and virion morphogenesis. In Aim 3, representatives from selected complementation groups in the combined Dales-Condit collection will be subjected to detailed analysis for defects in virion morphogenesis. Characterization will include assays for virion temperature sensitivity, post-translational cleavage of virion proteins, DNA packaging, synthesis, virion localization and intracellular localization of the protein product of the affected gene, detailed electron microscopic analysis of cells infected under non-permissive conditions, and biochemical analysis of accumulated aberrant intermediate particles. The successful completion of this project will enhance our understanding of the functional organization of the poxvirus genome, it will provide for the scientific community an extensive permanent collection of vaccinia virus conditional lethal mutants for future use in poxvirus functional genomics, and it will elucidate a genetic and biochemical pathway for virus morphogenesis. Historically, studies of virus morphogenesis have impacted significantly on our understanding of self assembly systems, membrane biology, intracellular protein trafficking, and virus cell interactions. We fully expect that our studies will have a similar impact. Importantly, the value of this type of research in basic poxvirology to public health has been significantly increased recently, given the potential for use of smallpox as a bioterrorist weapon.